Triiodothyronine Stimulates Specifically Growth Hormone Mrna
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Proc. Nati. Acad. Sci. U.SA Vol. 74, No. 5, pp. 2054-20581, May 1977 Cell Biology Triiodothyronine stimulates specifically growth hormone mRNA in rat pituitary tumor cells* (thyroid hormone/somatotropin/translation in cell-free system/mechanism of hormone action) HISAO SEOt, GILBERT VASSARTt, HUGETTE BROCASt, AND SAMUEL REFETOFFt t Thyroid Study Unit, University of Chicago, School of Medicine, Chicago, Illinois 60637; and * Institute de Recherche Interdisciplinaire, School of Medicine, Free University of Brussels, 1000 Brussels, Belgium Communicated by Elwood V. Jensen, March 10, 1977 ABSTRACT In a cell-free protein-synthesizing system from the T3-stimulated GH but not prolactin synthesis is accompa- a rabbit reticulocyte lysate, total RNA extracted from cultured nied by a specific increase in GH mRNA but not prolactin rat pituitary tumor (GH3) cells directed, in a dose-related man- mRNA activity. Thus, the known effect of T3 on GH production ner, the synthesis of proteins that were precipitated by antisera specific to rat growth hormone (somatotropin) and rat prolactin. may predominantly involve a selective stimulation of tran- A marked decrease in growth hormone secretion and growth scriptional activity. hormone mRNA activity was observed when cells were grown in a medium deficient in thyroid hormone. Addition of tri- METHODS iodothyronine in physiologic amounts both prevented and completely reversed this effect within 48 hr. Thyroid hormone Cell Culture. Rat pituitary tumor cells originating from the had no effect on prolactin secretion or prolactin mRNA activity. GH3 line were a gift from A. H. Tashjian. This line, derived These data suggest that thyroid hormone may stimulate syn- and thesis of growth hormone throug induction of transcriptional from a single MtTW5 rat pituitary tumor cell, synthesizes activity. The possibility of an additional effect at the posttran- secretes both GH and PRL (10). Except for the medium used, scriptional level has not been excluded. Although thyroid hor- the cells were propagated in culture as previously described mone is believed to have a general effect on a variety of meta- (11). Cells were maintained in modified Eagle's medium bolic processes, some effects, at the molecular level, may be (MEM) containing 10% fetal calf serum. For the experiments, quite selective, as indicated by the observed changes in growth cells were grown in MEM supplemented with 10% serum from hormone but not prolactin mRNA activity. The GH3 cell model is useful in the study of triiodothyronine action because of in- either normal (N) or thyroidectomized (Tx) rats, with or without dependence from secondary hormonal effects caused by hy- added T3. Each rat received 100,uCi of Na131I on the second pothyroidism and because simultaneous measurement of pro- day following surgical thyroidectomy and serum was harvested lactin mRNA activity serves as a unique internal control. 4 weeks later. Concentrations of endogenous thyroxine and T3 in the normal rat serum were 5.4 ,ug/100 ml and 60 ng/100 ml, The demonstration of triiodothyronine (T3) binding to nuclear respectively, and in the Tx rat serum were 0.6 ,g/100 ml and proteins raises the possibility that thyroid hormone may regulate less than 20 ng/100 ml, respectively. gene expression. Earlier work from Tata's laboratory showed RNA Extraction. Each RNA preparation was obtained from that thyroid hormone-induced protein synthesis was preceded 5 to 15 petri dishes or 30 to 70 X 106 cells treated in the same by formation of new RNA (1). Later, DeGroot et al. and Dil- manner. The medium was removed and saved for hormone man et al. showed increase in the poly(A)-rich fraction of RNA determination. The cells adhering to the bottom of the petri (2, 3). Demonstrations of stimulation of a specific mRNA by dishes were washed once with 3 ml of MEM, then harvested by thyroid hormones were recently provided by Kurtz et al. (4) gentle resuspension in the same medium, using a pasteur pi- and by Roy et al. (5) for a2u-globulin in the rat. However, be- pette. A 1 ml aliquot of the pooled 10 ml cell suspension was cause a number of hormones are known to stimulate the syn- saved for total RNA (12) and DNA (13) determination and the thesis of this protein (6) and because thyroid hormone pro- remainder was centrifuged for 10 min at 600 X g. The cell foundly alters the level of such hormones (7), experiments done pellet was resuspended in 1 ml of buffer containing 25 mM in the whole animal do not provide sufficient evidence for the MgCl2/50 mM KCI/200 mM sucrose/200 mM Tris-HCl, pH direct induction of -d2u-globulin mRNA by thyroid hormone. 8.5. In a rapid succession 7 ml of buffer containing 1% sodium Samuels et al. have reported a quantitative correlation be- dodecyl sulfate/100 mM NaCl/5 mM EDTA/0.02% hepa- tween nuclear T3 receptor occupancy and stimulation of growth rin/10 mM Tris, pH 8.5, was added, followed by 8 ml of phe- hormone (GH, somatotropin) synthesis in cultured rat pituitary nol/chloroform (1:1 vol/vol) presaturated with a buffer con- cells (8). We have adopted a similar experimental model to taining 100 mM NaCl/1 mM EDTA/10 mM Tris. Extraction study more directly the action of thyroid hormone on the in- was carried out by manual agitation for 5 min. Following duction of a specific mRNA. In a recent report, we have shown centrifugation for 10 min at 9000 X g the aqueous phase was that GH and prolactin mRNA activities in the total RNA ex- removed, and the organic phase containing the denatured tracted from a rat pituitary cell line (GH3) that actively syn- protein interphase was re-extracted after addition of 8 ml of the thesizes both hormones can be quantitated using a rabbit re- suspension buffer. The two aqueous phases were pooled and ticulocyte lysate cell-free system (9). In this report, we show that extracted four consecutive times with an equal volume of phenol/chloroform. During the final extraction no denatured Abbreviations: T3, triiodothyronine; GH, growth hormone (somato- protein interphase was present. Residual phenol in the aqueous tropin); Tx, thyroidectomized; N, normal; NRS, normal rabbit serum; phase was removed by a single extraction with chloroform, NMS, normal monkey serum; MEM, modified Eagle's medium. was added to a final concentration of 2 * Presented in part at the Fifty-Second Meeting of the American following which LiCl Thyroid Association, Inc., Toronto, Canada, September 15-18, M. The RNA was allowed to precipitate at 4° overnight. After 1976. centrifugation for 10 min at 9000 X g the RNA pellet was 2054 Downloaded by guest on September 26, 2021 Cell Biology: Seo, et al. Proc. Nati. Acad. Sci. USA 74 (1977) 2055 Table 1. Radioactivity in O-RNA and in immunologic blanks Radioactivity RNA, Specific antiserum Normal Anti-IgG serum in precipitate, Aug/ml (1st antibody)* carrier serumt (2nd antibody)t mean cpm ± range 0 Monkey anti-rat GH Goat anti-monkey IgG 1813 ± 187 o NMS Goat anti-monkey IgG 1953 ± 17 200 NMS Goat anti-monkey IgG 1933 ± 33 0 Rabbit anti-rat prolactin Goat anti-rabbit IgG 913 ± 13 0 NRS Goat anti-rabbit IgG 1058 ± 167 200 NRS Goat anti-rabbit IgG 960 ± 16 For details in preparation, see Methods. Volume and dilution of sera added to 200 or 250 ,. oflysate are given in the footnotes. Acid-precipitable radioactivity was 16.76 X 106 cpm. * Monkey anti-rat GH: 5 Al (1:50); rabbit anti-rat prolactin: 5 pl (1:25). t NMS = normal monkey serum: 5 Ml (1:50); NRS = normal rabbit serum: 5 Ml (1:5). 1 Goat anti-monkey IgG: 25 Al (undiluted); goat anti-rabbit IgG: 5 Al (undiluted). washed two times with cold 2 M LiCl followed by a single wash out on separate aliquots of lysate or on the same lysate in se- with 66% (vol/vol) ethanol in 0.3 M NaCl. The RNA was dis- quence. Either method achieved excellent recovery and re- solved in water and absorbance was read at 260 and 280 nm. producibility (Table 2). The specificity of the antibodies was The ratio in all preparations ranged from 1.9 to 2.1. The amount evaluated by their ability to precipitate purified authentic rat of RNA was calculated assuming that 1 A260 = 40,ug. After an GH and prolactin labeled with 125I. The antiprolactin serum ethanol precipitation, the final RNA preparation was dissolved was specific, and cross reaction of the anti-GH serum with in water to the desired concentretion to be used for transla- prolactin was eliminated by addition of 1 ,ug of prolactin tion. without affecting its reactivity with GH (Fig. 1). Furthermore, Cell-Free Protein Synthesis. Translation was carried out in as previously shown by comigration of the immunoprecipitates a rabbit reticulocyte lysate cell-free system as described by of authentic hormones and cell-free translated material on so- Palmiter et al. (14). After incubation for 1 hr at room temper- dium dodecyl sulfate/polyacrylamide gel electrophoresis (9), ature with RNA and [3H]leucine (Amersham, 53 Ci/mmol) at 95% of the radioactivity appeared as sharp single peaks. In 170 MCi/ml, the reaction was stopped by the addition of 0.1 accordance with findings by Sussman et al. (17), Evans et al. ml/ml of reaction mixture of a solution containing the following (18), and Maurer et al. (19), the hormones translated in the additives and bringing their concentrations to 0.02 M sodium cell-free system were of slightly larger molecular weights than phosphate buffer and 0.15 M NaCl, 1% (vol/vol) Triton X-100, the authentic hormones.